Antitumoral treatments

ABSTRACT

Aplidine and aplidine analogues are of use for the treatment of cancer, in particular in the treatment of leukemias and lymphomas, especially in combination therapies.

The present invention relates to combinations of aplidine or aplidineanalogues with other antitumoral agents, and the use of thesecombinations in the treatment of cancer, in particular in the treatmentof leukemias and lymphomas.

BACKGROUND OF THE INVENTION

Aplidine (Dehydrodidemnin B) is a cyclic depsipeptide that was isolatedfrom a Mediterranean marine tunicate, Aplidium albicans, and it is thesubject of WO 9109485. It is related to compounds known as didemnins,and has the following structure:

More information on aplidine, aplidine analogues, their uses,formulations and synthesis can be found in patent applications WO 981352, WO 99 42125, WO 01 76616, WO 01 35974, WO 02 30441 and WO 0202596. We incorporate by specific reference the content of each of thesePCT texts.

In both animal and human preclinical studies and in clinical Phase Istudies this agent has been shown to have cytotoxic potential against abroad spectrum of tumor types including leukemia and lymphoma. See forexample:

-   Faircloth, G. et al.: “Dehydrodidemnin B (DDB) a new marine derived    anticancer agent with activity against experimental tumour models”,    9th NCI-EORTC Symp New Drugs Cancer Ther (March 12-15, Amsterdam)    1996, Abst 111;-   Faircloth, G. et al.: “Preclinical characterization of aplidine, a    new marine anticancer depsipeptide”, Proc Amer Assoc Cancer Res    1997, 38: Abst 692;-   Depenbrock H, Peter R, Faircloth G T, Manzanares I, Jimeno J,    Hanauske A R.: “In vitro activity of Aplidine, a new marine-derived    anti-cancer compound, on freshly explanted clonogenic human tumour    cells and haematopoietic precursor cells” Br. J. Cancer, 1998; 78:    739-744;-   Faircloth G, Grant W, Nam S, Jimeno J, Manzanares I, Rinehart K.:    “Schedule-dependency of Aplidine, a marine depsipeptide with    antitumor activity”', Proc. Am. Assoc. Cancer Res. 1999; 40: 394;-   Broggini M, Marchini S, D'Incalci M, Taraboletti G, Giavazzi R,    Faircloth G, Jimeno J.: “Aplidine blocks VEGF secretion and    VEGF/VEGF-R1 autocrine loop in a human leukemic cell line”, Clin    Cancer Res 2000; 6 (suppl): 4509;-   Erba E, Bassano L, Di Liberti G, Muradore I, Chiorino G, Ubezio P,    Vignati S, Codegoni A, Desiderio M A, Faircloth G, Jimeno J and    D'Incalci M.: “Cell cycle phase perturbations and apoptosis in    tumour cells induced by aplidine”, Br J Cancer 2002; 86: 1510-1517;-   Paz-Ares L, Anthony A, Pronk L, Twelves C, Alonso S, Cortes-Funes H,    Celli N, Gomez C, Lopez-Lazaro L, Guzman C, Jimeno J, Kaye S.:    “Phase I clinical and pharmacokinetic study of aplidine, a new    marine didemnin, administered as 24-hour infusion weekly” Clin.    Cancer Res. 2000; 6 (suppl): 4509;-   Raymond E, Ady-Vago N, Baudin E, Ribrag V, Faivre S, Lecot F, Wright    T, Lopez Lazaro L, Guzman C, Jimeno J, Ducreux M, Le Chevalier T,    Armand J P.: “A phase I and pharmacokinetic study of aplidine given    as a 24-hour continuous infusion every other week in patients with    solid tumor and lymphoma”, Clin. Cancer Res. 2000; 6 (suppl): 4510;-   Maroun J, Belanger K, Seymour L, Soulieres D, Charpentier D, Goel R,    Stewart D, Tomiak E, Jimeno J, Matthews S.:“Phase I study of    aplidine in a 5 day bolus q 3 weeks in patients with solid tumors    and lymphomas”, Clin. Cancer Res. 2000; 6 (suppl): 4509;-   Izquierdo M A, Bowman A, Martinez M, Cicchella B, Jimeno J, Guzman    C, Germa J, Smyth J.: “Phase I trial of Aplidine given as a 1 hour    intravenous weekly infusion in patients with advanced solid tumors    and lymphoma”, Clin. Cancer Res. 2000; 6 (suppl): 4509.

Mechanistic studies indicate that aplidine can block VEGF secretion inALL-MOLT4 cells and in vitro cytotoxic activity at low concentrations(5nM) has been observed in AML and ALL samples from pediatric patientswith de novo or relapsed ALL and AML. Aplidine appears to induce both aG1, and a G2 arrest in drug treated leukemia cells in vitro. Apart fromdown regulation of the VEGF receptor, little else is known about themode(s) of action of aplidine.

In phase I clinical studies with aplidine, L-carnitine was given as a 24hour pretreatment or co-administered to prevent myelotoxicity, see forexample WO 02 30441. Co-administration of L-carnitine was proven to beable to improve the recovery of the drug induced muscular toxicity andhas allowed for dose escalation of aplidine.

Thus in clinical Phase I studies aplidine was not myelotoxic at maximumtolerated doses, except for mild lymphopenia. These characteristics makeaplidine a potentially useful agent for the treatment of leukemia.Adding aplidine to the current chemotherapy for leukemia could improveefficacy without the necessity of dose reductions of drugs with provenantileukemic activity, because of increased myelotoxicity. This seemsespecially relevant for the treatment of relapsed ALL and newlydiagnosed and relapsed AML, since these are diseases with a relativelypoor prognosis, which are currently being treated with myelotoxic drugcombinations.

SUMMARY OF THE INVENTION

We have for the first time established that aplidine and aplidineanalogues potentiate other anticancer agents and therefore can besuccessfully used in combination therapy for the treatment of cancer.This invention is directed to pharmaceutical compositions,pharmaceutical dosage forms, kits and methods for the treatment ofcancer using these combination therapies.

In accordance with one aspect of this invention, we provide effectivecombination therapies based on aplidine and aplidine analogues, usingother drugs which are effective in the treatment of cancer. Preferablythe other drug is effective in the treatment of leukemia and/orlymphoma. Most preferably the other drug is selected from the groupconsisting of methotrexate, cytosine arabinoside, mitoxantrone,vinblastine, methylprednisolone and doxorubicin.

In another embodiment the invention encompasses a method of treatingprimary and/or metastatic cancer comprising administering to a patientin need of such treatment a therapeutically effective amount of aplidineor an aplidine analogue, or a pharmaceutically acceptable prodrug, salt,solvate or hydrate thereof, and a therapeutically effective amount ofanother drug which is effective in the treatment of cancer or apharmaceutically acceptable prodrug, salt, solvate or hydrate thereof,administered prior, during, or after administering aplidine or aplidineanalogue.

Preferably the other drug is effective in the treatment of leukemiaand/or lymphoma. Most preferably the other drug is selected from thegroup consisting of methotrexate, cytosine arabinoside, mitoxantrone,vinblastine, methylprednisolone and doxorubicin. The other drugs mayform part of the same composition, or be provided as a separatecomposition for administration at the same time or at a different time.

The cancer to be treated is preferably a leukemia or a lymphoma, mostpreferably ALL, AML, CML, MML or CLL.

In another aspect the invention encompasses a method of increasing thetherapeutic efficacy of a drug effective in the treament of cancer,preferably a drug effective in the treatment of leukemia and/orlymphoma, most preferably a drug selected from the group consisting ofmethotrexate, cytosine arabinoside, mitoxantrone, vinblastine,methylprednisolone and doxorubicin, or a pharmaceutically acceptableprodrug, salt, solvate or hydrate thereof, which comprises administeringto a patient in need thereof an amount of aplidine or an aplidineanalogue, or a pharmaceutically acceptable prodrug, salt, solvate orhydrate thereof. Aplidine or the aplidine analogue is administeredprior, during, or after administering the other drug.

Aplidine or an aplidine analogue is able to increase the therapeuticefficacy of some cancer drugs. In one aspect, the result is synergism,rather than additive. Such synergistic combinations represent apreferred aspect of the present invention. Synergism may be indicated byuse of the Chou-Talalay method, or other methods. In other instances,antagonism may be found.

In a further aspect the invention encompasses a pharmaceuticalcomposition comprising aplidine or an aplidine analogue, or apharmaceutically acceptable prodrug, salt, solvate or hydrate thereof,and another drug effective in the treatment of cancer. Preferably theother drug is effective in the treatment of leukemia and/or lymphoma.Most preferably the other drug is selected from the group consisting ofmethotrexate, cytosine arabinoside, mitoxantrone, vinblastine,methylprednisolone and doxorubicin.

The invention also encompasses a kit for use in the treatment orprevention of cancer which comprises a dosage form of aplidine or anaplidine analogue, or a pharmaceutically acceptable prodrug, salt,solvate or hydrate thereof, a dosage form of another drug effective inthe treatment of cancer, or a pharmaceutically acceptable prodrug, salt,solvate or hydrate thereof, and instructions for the use of each actorin combination for the treatment or prevention of cancer. Preferably theother drug is effective in the treatment of leukemia and/or lymphoma.Most preferably the other drug is selected from the group consisting ofmethotrexate, cytosine arabinoside, mitoxantrone, vinblastine,methylprednisolone and doxorubicin.

In a further aspect, the invention is directed to the use of aplidinefor the treatment of chronic lymphocytic leukemia.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Aplidine inhibits growth of CLL cells in culture

FIG. 2. Aplidine is a potent inhibitor of preB-ALL cells in culture

FIG. 3. The cytotoxic dose-response curve of CCRF-CEM (FIG. 3A),SKI-DLCL (FIG. 3B) and K562 (3C) cells following aplidine treatment for96 hours

FIG. 4. Chou-Talalay analysis of combination of aplidine and AraC inCCRF-CEM cells

FIG. 5. Chou-Talalay analysis of combination of aplidine and AraC inSKI-DLCL cells

FIG. 6. Chou-Talalay analysis of combination of aplidine andmitoxantrone in CCRF-CEM cells

FIG. 7. Chou-Talalay analysis of combination of aplidine andmitoxantrone in SKI-DLCL cells

FIG. 8. Chou-Talalay analysis of combination of aplidine andmethotrexate in CCRF-CEM cells

FIG. 9. Chou-Talalay analysis of combination of aplidine and doxorubicinin CCRF-CEM cells

FIG. 10. Chou-Talalay analysis of combination of aplidine andvinblastine in CCRF-CEM cells

FIG. 11. Chou-Talalay analysis of combination of aplidine anddoxorubicin in SKI-DLCL cells

FIG. 12. Chou-Talalay analysis of combination of aplidine andvinblastine in SKI-DLCL cells

FIG. 13. Chou-Talalay analysis of combination of aplidine andmethylprednisolone in SKI-DLCL cells

FIG. 14. Combination of IC₂₀ of aplidine lowered the IC₅₀ of AraC inCCRF-CEM (FIG. 12A) and SKI-DLCL (FIG. 12B) cells after incubation for96 hours

FIG. 15. The effect of aplidine on in vivo tumor size as a single agentand in combination with AraC

DETAILED DESCRIPTION OF THE INVENTION

By cancer it is meant to include tumors, neoplasias, and any othermalignant tissue or cells. The present invention is directed to the useof aplidine or an aplidine analogue in combination for the treatments ofcancer in general, but more preferably for the treatment of differentleukemias and lymphomas.

In order to study the possible potentiation of other anticancer agentswith aplidine we have initiated a systematic study of drug combinationsfor possible use in leukemias and lymphomas. Aplidine was found to be aneffective in vitro cytotoxic agent against primary cells from a patientwith preB-ALL (DM4) as well as against fresh cells obtained from sixchronic lymphocytic leukemia (CLL) patients. The IC₅₀ value was 10 nMfor 3 day exposure with the DM4 line and after a 11 day exposure withthe primary CLL samples.

Drug combination studies were carried out on established cell linesrather than primary cells. We studied three cell lines viz. K562,CCRF-CEM and SKI-DLCL representing acute myeloid leukemia, lymphoblasticlymphoma and diffuse B cell large cell lymphoma respectively. The datain the examples show that Aplidine potentiates the effect ofmethotrexate, cytosine arabino side, mitoxantrone, vinblastine,methylprednisolone as well as doxorubicin in K562, CCRF-CEM and SKI-DLCLcells by lowering the IC₅₀s for the drugs.

Thus we have found that aplidine is a potent cytotoxic agent againstcells of several hematologic mailgnancies. Significantly, we haveestablished for the first time that aplidine inhibits growth of CLLcells in culture. We also found that aplidine enhances the cytotoxicityof agents used in the treatment of leukemias, such as methotrexate(MTX), cytosine arabonoside (AraC), mitoxantrone (Mitox), vinblastine(Vinb), methylprednisolone (Metpred) and doxorubicin (DOX).

Leukemia is classified by how quickly it progresses. Acute leukemia isfast-growing and can overrun the body within a few weeks or months. Bycontrast, chronic leukemia is slow-growing and progressively worsensover years.

The blood-forming (hematopoietic) cells of acute leukemia remain in animmature state, so they reproduce and accumulate very rapidly.Therefore, acute leukemia needs to be treated immediately, otherwise thedisease may be fatal within a few months. Fortunately, some subtypes ofacute leukemia respond to available therapies and they are curable.Children often develop acute forms of leukemia, which are manageddifferently from leukemia in adults.

In chronic leukemia, the blood-forming cells eventually mature, ordifferentiate, but they are not “normal”. They remain in the bloodstreammuch longer than normal white blood cells, and they are unable to combatinfection well.

Leukemia also is classified according to the type of white blood cellthat is multiplying—that is, lymphocytes (immune system cells),granulocytes (bacteria-destroying cells), or monocytes(macrophage-forming cells). If the abnormal white blood cells areprimarily granulocytes or monocytes, the leukemia is categorized asmyelogenous, or myeloid, leukemia. On the other hand, if the abnormalblood cells arise from bone marrow lymphocytes, the cancer is calledlymphocytic leukemia.

Other cancers, known as lymphomas, develop from lymphocytes within thelymph nodes, spleen, and other organs. Such cancers do not originate inthe bone marrow and have a biological behavior that is different fromlymphocytic leukemia.

There are over a dozen different types of leukemia, but four types occurmost frequently. These classifications are based upon whether theleukemia is acute versus chronic and myelogenous versus lymphocytic,that is:

Acute myelogenous leukemia (AML): also known as acute nonlymphocyticleukemia (ANLL)—is the most common form of adult leukemia. Most patientsare of retirement age (average age at diagnosis=65 years), and more menare affected than women. Fortunately, because of recent advances intreatment, AML can be kept in remission (lessening of the disease) inapproximately 60% to 70% of adults who undergo appropriate therapy.Initial response rates are approximately 65-75% but the overall curerates are more on the order of 40-50%.

Chronic myelogenous leukemia (CML) is known as a myeloproliferativedisorder—that is, it is a disease in which bone marrow cells proliferate(multiply) outside of the bone marrow tissue. CML is easy to diagnose,since it has a genetic peculiarity, or marker, that is readilyidentifiable under a microscope. About 95% of CML patients have agenetic translocation between chromosomes 9 and 22 in their leukemiccells. The Philadelphia chromosome causes uncontrolled reproduction andproliferation of all types of white blood cells and platelets (bloodclotting factors). CML is not yet curable by standard methods ofchemotherapy or immunotherapy.

Acute lymphocytic leukemia (ALL)—also known as acute lymphoblasticleukemia—is a malignant disease caused by the abnormal growth anddevelopment of early nongranular white blood cells, or lymphocytes. Theleukemia originates in the blast cells of the bone marrow (B-cells),thymus (T-cells), and lymph nodes. ALL occurs predominantly in children,peaking at 4 years of age.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in NorthAmerica and in Europe. It is a disease of older adults and is very rareamong people who are younger than 50 years of age. Men with CLLoutnumber women by a 2-to-1 average. CLL is thought to result from thegradual accumulation of mature, long-lived lymphocytes. Therefore, thiscancer is caused not so much by overgrowth as it is by the extremelongevity and build-up of malignant cells. Although the rate ofaccumulation varies among individuals, the extensive tumor burdeneventually causes complications in all CLL patients.

The compositions of the present invention may comprise both components(drugs) in a single pharmaceutically acceptable formulation.Alternatively, the components may be formulated separately andadministered in combination with one another. Various pharmaceuticallyacceptable formulations well known to those of skill in the art can beused in the present invention. Selection of an appropriate formulationfor use in the present invention can be performed routinely by thoseskilled in the art based upon the mode of administration and thesolubility characteristics of the components of the composition.

Examples of pharmaceutical compositions containing Aplidine or anaplidine analogue include liquid (solutions, suspensions or emulsions)with suitable composition for intravenous administration, and they maycontain the pure compound or in combination with any carrier or otherpharmacologically active compounds. Solubilised aplidine showssubstantial degradation under heat and light stress testing conditions,and a lyophilised dosage form was developed, see WO99/42125 incorporatedherein by reference.

Administration of aplidine or compositions of the present invention isbased on a Dosing Protocol preferably by intravenous infusion. We preferthat infusion times of up to 72 hours are used, more preferably 1 to 24hours, with about 1, about 3 or about 24 hours most preferred. Shortinfusion times which allow treatment to be carried out without anovernight stay in hospital are especially desirable. However, infusionmay be around 24 hours or even longer if required. Infusion may becarried out at suitable intervals with varying patterns, illustrativelyonce a week, twice a week, or more frequently per week, repeated eachweek optionally with gaps of typically one week.

The correct dosage of the compounds of the combination will varyaccording to the particular formulation, the mode of application and theparticular situs, host and tumour being treated. Other factors like age,body weight, sex, diet, time of administration, rate of excretion,condition of the host, drug combinations, reaction sensitivities andseverity of the disease shall be taken into account. Administration canbe carried out continuously or periodically within the maximum tolerateddose. Further guidance for the administration of aplidine is given in WO0135974 which is incorporated herein by reference in its entirety.

For the present invention, analogues of aplidine can be used in place ofAPL, aplidine itself. Typically such compounds are as defined in WO0202596. Examples of compounds for the present invention include thepreferred compounds given in WO 0202596, and in particular we importinto this patent specification the discussion of preferred compounds andrelated aspects given in WO 0202596. More preferably, the analogues arestructurally close to aplidine, and usually differ from aplidine inrespect of one amino acid or the terminal sidechain. The different aminoacid can be in the cyclic part of the molecule or in the sidechain. Manyexamples of such compounds are given in WO 0202596, and they arecandidates for use in the present invention.

EXAMPLE Example 1

Aplidine was tested against various primary cells from patients withhematologic malignancies. The cells used were:

-   fresh cells obtained from six chronic lymphocytic leukemia patients-   primary cell from a patient with preB-ALL (DM4)

Patient samples were obtained with prior consent and CLL cells wereisolated by density gradient centrifugation over histopaque. The mediaused was RPMI supplemented with 10% autologous serum and L-glutamine.The cultures were incubated with 10 nM aplidine and cell viability wasmeasured days 3, 7,11 and 18 and compared with viability of untreatedcells and STI 571 (0.5 mM).

The results of these studies are shown in FIGS. 1-2.

Example 2

In order to study the possible potentiation of other anticancer agentswe undertook a study of drug combinations for possible use in leukemiasand lymphomas.

Drug combination studies were carried out on established cell linesrather than primary cells. We studied three cell lines, viz. K562 as amodel for acute myeloid leukemia, CEM representing acute lymphocyticleukemia and SKI-DLCL representing diffuse large cell lymphoma.Combination studies with IC20 and IC 50 dose of aplidine with a doserange of methotrexate, cytosine arabinoside and doxorubicin were testedto determine if aplidine could potentiate the effect of these drugs.

The results are shown in table 1: Additional Drug IC50 Dox IC50 MTX IC50Ara-C No Aplidine 18 nM 5 nM 30 nM IC₂₀ Aplidine (0.5 nM) 1 nM 500 pM 6nMp < 0.01, p < 0.05, p < 0.05

Clearly, these data show that aplidine potentiates the effect ofdoxorubicin, methotrexate and cytosine arabinoside by lowering verysignificantly the IC₅₀s for the drugs.

Example 3 In vitro Studies to Determine the Effect of Aplidine as aSingle Agent on CCRF-CEM, SKI-DLCL and K562 Cell Lines

CCRF-CEMS, SKI-DLCL and K562 cells are maintained in RPMI 1640supplemented with 10% FCS. To determine the cytotoxic effect of aplidineon all cell lines and to obtain the IC₅₀ of aplidine in these celllines, cells were plated into 96 well plates and incubated for 96 hoursin humidified and 5% CO2 containing incubator. Cell viability ismeasured by XTT assay in an automated plate reader. We found aplidine tobe cytotoxic to all cell lines with an IC₅₀ dose of 0.5-1.0 nM (FIG. 3).

Example 4 Studies on in vitro Effect of Aplidine+Drug Combination withFixed Doses of IC₅₀:IC₅₀ on All Cell Lines

Methotrexate, cytosine arabinoside C (ara-C), mitoxantrone,methylprednisolone, vinblastine and doxorubicin were tested incombination with aplidine.

Chou-Talalay analysis was used to analyze the drug combinations. WhenCombination Index (CI) obtained by this analysis is less than 1, thedrugs are synergistic; when CI is 1, the drugs are additive; and, if CIis greater than 1, the drugs are antagonistic.

All the citotoxicity studies were performed by using XTT or MTS. Wefirst determined the IC₅₀ dose of these drugs in SKI-DLCL, CCRF-CEM andK562 cell lines. We investigated drug combinations usingIC₅₀(Aplidine):IC₅₀(DrugX) fixed ratio.

In table 2 is shown the combination of aplidine and Ara-C with the doseof (IC₅₀:IC₅₀) in CCRF-CEM cells. TABLE 2 Viability Ratio Dose of APLDose of AraC (% of control) Control 0 0 100 IC50(APL) 0.5 nM 0 52.7IC50(AraC) 0 10 nM 56.4 ×16 8 nM 160 nM 4.7 ×8 4 nM 80 nM 7.9 ×4 2 nM 40nM 7.6 ×2 1 nM 20 nM 7.8 IC50:IC50 0.5 nM 10 nM 10.6 ×½ 0.25 nM 5 nM16.2 ×¼ 1.125 nM 2.5 nM 36.7 ×⅛ 0.0625 nM 1.25 nM 70.8

The results of Chou-Talalay analysis of combination of aplidine andAra-C in CCRF-CEM cells can be seen in FIG. 4. The CI for thiscombination in CCRF-CEM cells is 0.469.

In table 3 is shown the combination of aplidine and Ara-C with the doseof (IC₅₀:IC₅₀) in SKI-DLCL cells. TABLE 3 Viability Ratio Dose of APLDose of AraC (% of control) Control 0 0 100 IC50(APL) 0.5 nM 0 50IC50(AraC) 0 30 nM 50 ×16 8 nM 480 nM 12 ×8 4 nM 240 nM 10.7 ×4 2 nM 120nM 14.1 ×2 1 nM 60 nM 17.4 IC50:IC50 0.5 nM 30 nM 23.1 ×½ 0.25 nM 15 nM25.4 ×¼ 1.125 nM 7.5 nM 25.5 ×⅛ 0.0625 nM 3.75 nM 50.8

The results of Chou-Talalay analysis of combination of aplidine andAra-C in SKI-DLCL cells can be seen in FIG. 5. The CI for thiscombination in SKI-DLCL cells is 0.306.

In table 4 is shown the combination of aplidine and Ara-C with the doseof (IC₅₀:IC₅₀) in K562 cells. Viability Ratio Dose of APL Dose of AraC(% of control) Control 0 0 100 IC50(APL) 1 nM 0 50 IC50(AraC) 0 30 nM 50×16 16 nM 480 nM 11.8 ×8 8 nM 240 nM 15.2 ×4 4 nM 120 nM 15.5 ×2 2 nM 60nM 17 IC50:IC50 1 nM 30 nM 22.1 ×½ 0.5 nM 15 nM 25.6 ×¼ 0.25 nM 7.5 nM31.1 ×⅛ 0.125 nM 3.75 nM 44.2

The CI for this combination in K562 cells is 0.502.

In table 5 is shown the combination of aplidine and mitoxantrone withthe dose of (IC₅₀:IC₅₀) in CCRF-CEM cells. Viability Ratio Dose of APLDose of Mitoxantrone (% of control) Control 0 0 100 IC50(APL) 0.5 nM 050 IC50(Mitox) 0 30 nM 56 ×16 8 nM 480 nM 9.9 ×8 4 nM 240 nM 11.6 ×4 2nM 120 nM 11.9 ×2 1 nM 60 nM 13.8 IC50:IC50 0.5 nM 30 nM 20.6 ×½ 0.25 nM15 nM 39.7 ×¼ 1.125 nM 7.5 nM 60.7 ×⅛ 0.0625 nM 3.75 nM 76.5

The results of Chou-Talalay analysis of combination of aplidine andmitoxantrone in CCRF-CEM cells can be seen in FIG. 6. The CI for thiscombination in CCRF-CEM cells is 0.911.

In table 6 is shown the combination of aplidine and mitoxantrone withthe dose of (IC₅₀:IC₅₀) in SKI-DLCL cells. Viability Dose of APL Dose ofMitoxantrone (% of control) Control 0 0 100 IC50(APL) 0.5 nM 0 50IC50(Mitox) 0 5 nM 50 ×16 8 nM 80 nM 17 ×8 4 nM 40 nM 29 ×4 2 nM 20 nM22.6 ×2 1 nM 10 nM 19.9 IC50:IC50 0.5 nM 5 nM 32.2 ×½ 0.25 nM 2.5 nM53.1 ×¼ 1.125 nM 1.25 nM 58.6 ×⅛ 0.0625 nM 0.625 nM 70.1

The results of Chou-Talalay analysis of combination of aplidine andmitoxantrone in SKI-DLCL cells can be seen in FIG. 7. The CI for thiscombination in SKI-DLCL cells is 0.646.

In table 7 is shown the combination of aplidine and mitoxantrone withthe dose of (IC₅₀:IC₅₀) in K562cells. Viability Dose of APL Dose ofMitoxantrone (% of control) Control 0 0 100 IC50(APL) 1 nM 0 50IC50(Mitox) 0 7.5 nM 50.7 ×16 16 nM 120 nM 9.9 ×8 8 nM 60 nM 11.6 ×4 4nM 30 nM 11.9 ×2 2 nM 15 nM 13.8 IC50:IC50 1 nM 7.5 nM 20.6 ×½ 0.5 nM3.75 nM 39.7 ×¼ 0.25 nM 1.8 nM 60.7 ×⅛ 0.125 nM 0.9 nM 76.5

The CI for this combination in K562 cells is 0.487.

In table 8 is shown the combination of alidine and mthotrexate with thedose of (IC₅₀:IC₅₀) in CCRF-CEM cells. Viability Ratio Dose of APL Doseof Metotrexate (% of control) Control 0 0 100 IC50(APL) 0.5 nM 0 50IC50(MTX) 0 10 nM 50 ×16 8 nM 160 nM 5 ×8 4 nM 80 nM 13 ×4 2 nM 40 nM 11×2 1 nM 20 nM 12 IC50:IC50 0.5 nM 10 nM 20 ×½ 0.25 nM 5 nM 30 ×¼ 1.125nM 2.5 nM 88 ×⅛ 0.0625 nM 1.25 nM 100

The results of Chou-Talalay analysis of combination of alidine andmthotrexate in CCRF-CEM cells can be seen in FIG. 8. The CI for thiscombination in CCRF-CEM cells is 0.950.

The results of Chou-Talalay analysis of combination of aplidine andDoxorubicin in CCRF-CEM cells can be seen in FIG. 9. The CI for thiscombination in CCRF-CEM cells is 1.952.

The results of Chou-Talalay analysis of combination of Aplidine andvnblastine in CCRF-CEM cells can be seen in FIG. 10. The CI for thiscombination in CCRF-CEM cells is 2.046.

In table 9 is shown the combination of alidine and dxorubicin with thedose of (IC₅₀:IC₅₀) in SKI-DLCL cells. Viability Ratio Dose of APL Doseof Doxorubicin (% of control) Control 0 0 100 IC50(APL) 0.5 nM 0 50IC50(Doxo) 0 5 nM 50 ×16 8 nM 80 nM 9.4 ×8 4 nM 40 nM 8.6 ×4 2 nM 20 nM8 ×2 1 nM 10 nM 9.7 IC50:IC50 0.5 nM 5 nM 21 ×½ 0.25 nM 2.5 nM 40 ×¼1.125 nM 1.25 nM 45 ×⅛ 0.0625 nM 0.62 nM 49

The results of Chou-Talalay analysis of combination of alidine anddxorubicin in SKI-DLCL cells can be seen in FIG. 11. The CI for thiscombination in SKI-DLCL cells is 0.478.

In table 10 is shown the combination of alidine and vnblastine with thedose of (IC₅₀:IC₅₀) in SKI-DLCL cells. Viability Ratio Dose of APL Doseof Vinblastine (% of control) Control 0 0 100 IC50(APL) 0.5 nM 0 50IC50(Vinb) 0 4 nM 50 ×16 8 nM 64 nM 15 ×8 4 nM 32 nM 17 ×4 2 nM 16 nM 17×2 1 nM 8 nM 21 IC50:IC50 0.5 nM 4 nM 29 ×½ 0.25 nM 2 nM 25 ×¼ 1.125 nM1 nM 28 ×⅛ 0.0625 nM 0.5 nM 38

The results of Chou-Talalay analysis of combination of alidine andvnblastine in SKI-DLCL cells can be seen in FIG. 12. The CI for thiscombination in SKI-DLCL cells is 0.760.

In table 11 is shown the combination of alidine and mthylprednisolonewith the dose of (IC₅₀:IC₅₀) in SKI-DLCL cells. Dose of Viability (% ofDose of APL methylprednisolone control) Control 0 0 100 IC50(APL) 0.5 nM0 50 IC50(Metpred) 0 160 nM 51 ×16 8 nM 2560 nM 10.8 ×8 4 nM 1280 nM17.3 ×4 2 nM 640 nM 16.7 ×2 1 nM 320 nM 17.4 IC50:IC50 0.5 nM 160 nM24.7 ×½ 0.25 nM 80 nM 32.4 ×¼ 1.125 nM 40 nM 39.1 ×⅛ 0.0625 nM 20 nM 50

The results of Chou-Talalay analysis of combination of alidine andmthylprednisolone in SKI-DLCL cells can be seen in FIG. 13. The CI forthis combination in SKI-DLCL cells is 0.646.

Example 5

We have also investigated the cytotoxic effect of combination of IC₂₀(APL) with a variable dose of AraC on CCRF-CEM and SKI-DLCL cell lines.Aplidine in both cell lines potentiated the effect of AraC, the IC₅₀dose of AraC was reduced from 30 nM to 1.6 nM in SKI-DLCL cell line, andfrom 10 nM to 0.8 nM in CCRF-CEM cell line respectively (FIG. 14). Datawas obtained after cell incubation for 96 hours and using XTT asay. Theresults represent means of three different experiments.

Example 6 In vivo Studies

We have performed in vivo experiments to study the effect of alidinealone and in combination with other drugs for lymphoid malignancies.

Determination of Maximum Tolerated Dose (MTD) in C.B.-17 scid/scid (SCIDMice)

We have used an in vivo model of human lymphoma in SCID mice for thispurpose. Specifically, we have used CCRF-CEMS cells and CB.17 scid/scidmice. We have experience with this model and have evaluated drugtreatments using this xenograft (Lacerda J. F. et al. Blood 85 (10):2675-2679 (1995)). We found that a total dose of 1 mg/kg/week given infive daily doses is the aplidine maximum dose that can be tolerated bymice.

Determination of in vivo Antitumor Effect of Alidine as a Single Agentand in Combination with AraC in SCID Mice Xenograft Model

SCID mice were inoculated subcutaneously in the right flank with 10⁷CEM-T leukemic cells. They were observed twice weekly for tumorformation at the site of inoculation. After establishment of palpabletumor, alidine was injected as single agent and in combination withseveral doses of AraC to determine the antitumor effect. Mice wererandomized to receive alidine alone at doses of 0.75 mg/kg and 1 mg/kg,AraC alone at 50 mg/kg, or combination of Aplidine and AraC for all dosecombinations. The AraC dose chosen for this combination is the dose atwhich the tumor growth was inhibited but no tumor regression occurred.All drugs were administered intra-peritoneally, and tumor size wascompared to a control group of mice not receiving any treatment and forcombination groups, compared to tumor sizes with single agent treatment.

The most effective combination was found to be AraC-50mg/kg+alidine-0.75 mg/kg (FIG. 15).

These findings in respect of aplidine can be extended to aplidineanalogues, derivatives and related compounds. For example, the presentinvention provides a combination of a compound such as those of WO 0202596 with an anticancer drug, preferably an anti-leukemia drug oranti-lymphoma drug, notably methotrexate, cytosine arabinoside,mitoxantrone, vinblastine, methylprednisolone or doxorubicin.

1. Use of aplidine or an aplidine analogue in the manufacture of amedicament for the treatment of primary and/or metastatic cancer bycombination therapy employing aplidine or an aplidine analogue withanother drug.
 2. The use of aplidine or an aplidine analogue accordingto claim 1, wherein the cancer to be treated is a leukemia or alymphoma.
 3. The use of aplidine or an aplidine analogue according toany of the preceding claims, wherein the aplidine or the aplidineanalogue and the other drug form part of the same medicament.
 4. The useof aplidine or an aplidine analogue according to any of claims 1 to 3wherein the aplidine or the aplidine analogue and the other drug areprovided as separate medicaments for administration at the same time orat different times.
 5. The use of aplidine or an aplidine analogueaccording to any of the preceding claims, wherein the other drug isselected from the group consisting of methotrexate, cytosinearabinoside, mitoxantrone, vinblastine, methylprednisolone anddoxorubicin.
 6. The use of aplidine or an aplidine analogue according toany of the preceding claims, wherein the aplidine or the aplidineanalogue is aplidine.
 7. A method of treating primary and/or metastaticcancer comprising administering to a patient in need of such treatment atherapeutically effective amount of aplidine or an analogue of aplidineand a therapeutically effective amount of another drug which iseffective in the treatment of cancer, administered prior, during, orafter administering the aplidine or the aplidine analogue.
 8. The methodof treating primary and/or metastatic cancer according to claim 6,wherein the cancer to be treated is a leukemia or a lymphoma.
 9. Themethod of treating primary and/or metastatic cancer according to claim 7or 8, wherein the other drug is selected from the group consisting ofmethotrexate, cytosine arabinoside, mitoxantrone, vinblastine,methylprednisolone and doxorubicin.
 10. A method of increasing thetherapeutic efficacy of a drug effective in the treatment of cancer,which comprises administering to a patient in need thereof an amount ofaplidine or an analogue of aplidine, the aplidine or the analogue ofaplidine being administered prior, during, or after administering theother drug.
 11. The method of increasing the therapeutic efficacy of adrug effective in the treatment of cancer according to claim 10, whereinthe cancer to be treated is a leukemia or a lymphoma.
 12. The method ofincreasing the therapeutic efficacy of a drug effective in the treatmentof cancer according to claims 10 or 11, wherein the drug is selectedfrom the group consisting of methotrexate, cytosine arabinoside,mitoxantrone, vinblastine, methylprednisolone and doxorubicin.
 13. Apharmaceutical composition comprising aplidine or an aplidine analogueand another drug effective in the treatment of cancer.
 14. Thepharmaceutical composition according to claim 13, wherein the other drugis effective in the treatment of a leukemia and/or a limphoma.
 15. Thepharmaceutical composition according to any of claims 13 or 14, whereinthe other drug is selected from the group consisting of methotrexate,cytosine arabinoside, mitoxantrone, vinblastine, methylprednisolone anddoxorubicin.
 16. A kit for use in the treatment or prevention of cancerwhich comprises a dosage form of aplidine or an aplidine analogue, adosage form of another drug effective in the treatment of cancer andinstructions for the use of each actor in combination for the treatmentor prevention of cancer.
 17. The kit according to claim 16, wherein thekit is for use in the treatment or prevention of a leukemia and/or alymphoma.
 18. The kit according to any of claims 16 or 17, wherein theother drug is selected form the group consisting of methotrexate,cytosine arabinoside, mitoxantrone, vinblastine, methylprednisolone anddoxorubicin.
 19. A use of aplidine or an aplidine analogue in themanufacture of a medicament for the treatment of chronic lymphocyticleukaemia.